<Supporting information>
+A: Supplementary materials and methods
+B: Acid-washed glass/zirconia beads
+C: Materials
0.5 mm glass beads (GB-05; Tomy Seiko Co. Ltd, Japan)
0.5 mm zirconia beads (ZSB-05; Tomy Seiko)
0.1 mm zirconia beads (ZSB-01; Tomy Seiko)
100 ml Pyrex bottle
200 ml glass beaker
Diethyl dicarbonate (048-21902; Wako, Japan)
Hydrochloric acid (087-01076; Wako)
5.8 M hydrochloric acid
Add 1 volume concentrated HCl (36%) to 1 volume distilled water with moderate
stirring.
+C: Method
1.
Weigh 50 g beads into a 100 ml Pyrex bottle.
2.
Add 5.8 M HCl to the 60 ml mark on the bottle.
3.
Incubate at room temperature overnight.
4.
Carefully decant 5.8 M HCl from the bottle to a 200 ml glass beaker and discard
according to your local regulations.
5.
Add distilled water to the 100 ml mark on the bottle and swirl bottle for 10 s to stir
up beads.
6.
Decant the distilled water wash directly over the drain and rinse wash volume down
the drain with tap water.
7.
Repeat steps 5 and 6 for a total of 10 washes to reduce the HCl concentration below
10 mM.
8.
To remove RNase, add distilled water to the 60 ml mark and add 6 µl diethyl
dicarbonate, swirl for 10 s and allow to stand for 1 h at room temperature.
9.
Decant the distilled water wash again directly over the drain and rinse wash volume
down the drain with tap water.
10.
Autoclave beads for 20 min at 121°C.
11.
Dry the beads in an oven at 75°C.
12.
Store acid-washed, sterile RNase-free beads at room temperature.
<Footnotes>
Be careful – HCl is a fuming substance; dilute in a fume hood if necessary.
1
2
The volume of beads should be no more than one-fifth of the volume of the bottle used for
washes. To scale up, use 100 g beads and a 250 ml Pyrex bottle.
3
Alternatively, use concentrated nitric acid.
4
Some HCl will remain with the beads.
+B: Extraction of Saccharomyces cerevisiae DNA using the TNE bead-beating method
+C: Materials
5 ml microcentrifuge tube (T2076S-C; Argo Technologies, USA)
1.5 ml screw-cap O-ring tube (T334-5SPR; Simport, Canada)
0.5 mm glass beads (GB-05; Tomy Seiko)
1.5 ml microcentrifuge tube snap cap (022364111; Eppendorf, Japan)
Sodium chloride (191-01665; Wako, Japan)
Tris base (204-07885; Wako)
Na2EDTA (345-01865; Dojindo, Japan)
RNase A solution 4 mg/ml (A7973; Promega, Japan)
Phenol:chloroform:isoamyl alcohol 25:24:1 (311-90151; Nippon Gene, Japan)
UltraPure™ distilled water (10977-015; Life Technologies, USA)
TNE buffer2 (pH 7; adjust pH using HCl)
58.44 g sodium chloride 1 M
12.11 g Tris base 100 mM
0.372 g Na2-EDTA (FW 372.24) 1 mM
Up to 1 l distilled water
+B: Method
1.
Quench 5 × 108 cells (1 ml) in two volumes of ethanol (2 ml; use 5 ml
microcentrifuge tube).
2.
Pellet the sample by centrifuging at 2000  g for 1 min and discard the supernatant.
3.
Resuspend the pellet in 300 µl TNE buffer and transfer to a 1.5 ml screw cap tube.
Optional: for RNAsing, add 3 µl RNAse A.
4.
Add the glass beads (~300 µl; 0.5 mm) and bead-beat using the following protocol:
3× (10 s beat at 5500 rpm; 30 s rest). Optional: for RNAsing, incubate at 37°C for
30 min with gentle shaking (300 rpm; MB-101, BIOER, Japan).
5.
Add one volume (300 µl) phenol:chloroform:isoamyl alcohol 25:24:1 and mix by
gentle inversion.
6.
Centrifuge at 12 000  g for 15 min.
7.
Transfer the aqueous phase into a fresh 1.5 ml microcentrifuge tube. Optional: for
back-extraction, add 0.5 volume TNE buffer (150 µl) to the phenol phase, gently
mix, repeat step 6 and combine the aqueous phases.
8.
An optional chloroform cleaning can be performed if the aqueous phase is
contaminated.
9.
Add two volumes ice-cold ethanol then gently mix on a rotor for 5 min (minimum
speed; BC-7101, BioCraft, Japan).
10.
Centrifuge at 12 000  g for 15 min and discard the supernatant.
11.
Wash the pellet twice using 500 µl 70% ethanol and air-dry for 5 min.
12.
Dissolve the pellet in 50–100 µl UltraPure™ distilled water or TE buffer, pH 8.
13.
In the short term (a few days), the sample can be stored at 4°C; for long-term
storage, lyophilize and store at –20°C.
<Footnotes>
1
Beads must be acid-washed prior to use [see standard operating procedure (SOP)].
2
Autoclave at 121°C for 20 min.
3
Unless stated otherwise, all the procedures are carried out at room temperature.
4
Resuspend and transfer using 200 µl TNE buffer first, then use 100 µl TNE buffer to wash and
transfer the remaining.
Use the refrigerated Micro Smash™ bead-beater (MS-100, Tomy Seiko).
5
+B: Extraction of Saccharomyces cerevisiae RNA using the sodium acetate bead-beating
method
+C: Materials
1.5 ml screw-cap O-ring tube (T334-5SPR; Simport, Canada)
0.5 mm zirconia beads (ZSB-05; Tomy Seiko)
0.1 mm zirconia beads (ZSB-01; Tomy Seiko)
1.5 ml microcentrifuge tube snap cap (022364111; Eppendorf)
Sodium acetate (192-01075; Wako)
Na2-EDTA (345-01865; Dojindo)
Diethyl dicarbonate (048-21902; Wako)
TE-saturated phenol (26829-96; Nacalai Tesque, Japan)
UltraPure™ distilled water (10977-015; Life Technologies, USA)
Sodium acetate buffer (pH 4.5–5.0; adjust pH using acetic acid)
24.61 g sodium acetate 300 mM
3.72 g Na2-EDTA (FW 372.24) 10 mM
1 ml diethyl dicarbonate 0.1% v/v
Up to 1 l UltraPure™ distilled water
+C: Method
1.
Quench 2.5 × 108 cells (500 µl) in two volumes ethanol (1 ml; use 1.5 ml screwcap tube, equilibrated to –70°C using a dry-ice bath). Stop point: samples can be
stored at –80°C overnight.
2.
Pellet the sample by centrifuging at 10 000  g for 1 min and discard the
supernatant.
3.
Resuspend the pellet in 250 µl sodium acetate buffer.
4.
Add one volume TE-saturated phenol equilibrated with sodium acetate buffer (250
µl).
5.
Add the zirconia bead mix (~300 µl; 1:1) and bead-beat using the following
protocol: 4 × [3× (10 s beat at 5500 rpm; 30 s rest); 120 s rest].
6.
Centrifuge at 12 000  g for 15 min.
7.
Transfer the aqueous phase into a fresh 1.5 ml microcentrifuge tube.
8.
For back-extraction, add a 0.5 volume sodium acetate buffer (125 µl) into the
phenol phase, vortex for 10 s, repeat step 6 and combine the aqueous phases.
9.
Add 2.5 volumes ice-cold ethanol to the aqueous phase and precipitate the
nucleotides by incubating at –20°C overnight.
10.
Centrifuge at 12 000  g for 30 min and discard the supernatant.
11.
Wash the pellet three times using 500 µl 70% ice-cold ethanol and air-dry for 10
min at room temperature.
12.
Dissolve the pellet in 50–100 µl UltraPure™ distilled water or 10 mM sodium
acetate (pH 4.5–5.0) and store at –80°C.
<Footnotes>
1
Beads must be acid-washed and RNAse-free prior to use (see SOP).
2
Incubate at room temperature for 1 h and autoclave at 121°C for 20 min.
3
Unless stated otherwise, all the procedures were carried out at 4°C.
Use the refrigerated Micro Smash™ bead-beater (MS-100, Tomy Seiko).
4
5
Treated with diethyl dicarbonate (0.1% v/v added and incubated for 1 h at room temperature) and
autoclaved at 121°C for 20 min.
+B: Extraction of Saccharomyces cerevisiae protein using the ethanol bead-beating
method
+C: Materials
1.5 ml screw-cap O-ring tube (T334-5SPR; Simport)
0.5 mm glass beads (GB-05; Tomy Seiko)
1.5 ml microcentrifuge tube snap cap (022364111; Eppendorf)
2 ml microcentrifuge tube snap cap (022363344; Eppendorf)
Tris-base (204-07885; Wako)
Hydrochloric acid (087-01076; Wako)
Sodium dodecylsulphate (191-07145; Wako)
Glycerol (075-00611; Wako)
Phosphate-buffered saline (PBS) 10, pH 7.4 (AM9625; Life Technologies)
UltraPure™ distilled water (10977-015; Life Technologies, USA)
2 Laemmli buffer
252 ml Tris–HCL 0.5 M, pH 6.8, 126 mM
400 ml SDS 10% w/v 4%
200 ml glycerol 20%
Up to 1 l distilled water
+C: Method
1.
Quench 2.5 × 108 cells (500 µl) in two volumes ethanol (1 ml; use 1.5 ml screwcap tube, equilibrated to –70°C using dry-ice bath).
2.
Pellet the sample by centrifuging at 10 000  g for 1 min and discard the
supernatant.
3.
Wash the pellet by resuspending in 1 ml 1 PBS, centrifuge at 10 000  g for 1 min
and discard the supernatant.
4.
Resuspend the pellet in 400 µl ice-cold ethanol.
5.
Add the glass beads (~300 µl; 0.5 mm) and bead-beat using the following protocol:
3× (30 s beat at 5500 rpm; 60 s rest).
6.
Invert the screw cap tube and drill a hole (0.5 mm) into the conical base.
7.
Place the pierced tube firmly in a fresh 2 ml microcentrifuge collection tube.
8.
Centrifuge at 5000  g for 5 min.
9.
For back-extraction, add 0.5 volume of 200 µl ice-cold ethanol into the pierced tube
and repeat step 8.
10.
Remove the pierced tube and incubate the collection tube at –20°C for 1 h.
11.
Centrifuge at 16 000  g for 15 min and remove the supernatant.
12.
Resuspend the pellet in 500 µl 1 Laemmli buffer and incubate at 95°C for 10 min
with gentle shaking (300 rpm; MB-101, BIOER, Japan).
13.
Incubate at room temperature for 1 min.
14.
Centrifuge at 16 000  g for 1 min and transfer the supernatant into a fresh
microcentrifuge tube.
15.
Store at –20°C (make aliquots if necessary).
<Footnotes>
1
Beads must be acid-washed prior to use (see SOP).
2
Unless stated otherwise, all the procedures were carried out at 4°C.
Use the refrigerated Micro Smash™ bead-beater (MS-100, Tomy Seiko).
3
4
Tap to move the lysate bead mixture from the bottom part of the tube.